CN108990793B - Breeding method of tetraploid petunia - Google Patents
Breeding method of tetraploid petunia Download PDFInfo
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Abstract
The invention discloses a method for cultivating quadruple petunia, which comprises the following steps: mutagenizing axillary buds of diploid petunia plants by using a chemical inducer, and identifying and screening tetraploid branches after the mutagenized axillary buds germinate and grow into branches; taking tetraploid petunia with good fertility as a female parent, emasculating, taking out mature 2n pollen on the tetraploid branches obtained by mutagenesis, coating the pollen on the female parent column, and obtaining F1 hybrid seeds after the plants are fruited; tetraploid plants are screened out in the F1 generation and are selected by a pedigree method, and the tetraploid petunia variety with excellent properties is obtained. The method can improve the mutagenic rate of the polyploidy, the cultured tetraploid variety has wider genetic diversity, and the method not only has some excellent properties of the diploid male parent and higher ornamental value, but also has higher seed setting rate, and simultaneously, the method has low requirements on operation fields and instruments and equipment, and the operation process is relatively simple and easy.
Description
Technical Field
The invention relates to a method for cultivating quadruple petunia, belonging to the field of horticultural plant genetic breeding.
Background
Petunia hybrida Vilm is a plant of the genus Petunia of the family solanaceae, an annual herb flower. Petunia flowers are large, rich in color and quite variable in flower types, and the petunia is a main potted flower and decorative plant internationally at present, is widely applied to beautification, decoration and greening of courtyards, open-air flower beds and squares, and is liked to the king of flower beds in the world. In China, petunia is introduced and cultivated in a small amount at the beginning of the 20 th century, and in the 80 th era, new species are gradually introduced from countries such as Europe and America for scale planting, so that the petunia is increasingly required along with economic development and urban construction.
The polyploid plant is characterized in that the somatic cell contains three or more chromosome groups, and the polyploid plant is widely applied due to the characteristics of high giant property, strong stress resistance, low fructification, high nutritional ingredients and the like, and the polyploid breeding has wide prospect in modern breeding. At present, the artificial induction of plant polyploidy mainly comprises two methods of asexual polyploidy and sexual polyploidy.
(1) Asexual polyploidization (Somatic polyploidization), chromosome doubling occurs during mitosis, i.e., the chromosome doubled Somatic cells develop into polyploid plants. Common induction modes are physical and chemical. Physical modes include temperature shock, mechanical trauma, ionizing radiation and the like; the chemical modes are mostly drug induction, mainly including colchicine, indoleacetic acid, naphthoethane, coriander, and the like. Colchicine is the most effective chemical inducer, and when it comes into contact with mitotic cells, it inhibits the microtubule polymerization process, and does not form spindle threads, which makes the chromosomes not lie on the equatorial plate and not move to the two poles of the cell, thus producing chromosome-doubled nuclei.
(2) Sexual polyploidization (Sexual polyploidization). Sexual polyploidization is relative to chromosome doubling in the asexual mitosis process, and the sexual polyploidy is formed by treating young buds with an inducer and inducing diploid meiosis disorder to generate 2n gametes to participate in hybrid fertilization in the meiotic stage of microsporocytes. Researches show that the 2n gamete plays a very important role in the plant evolution process and has special value in transmitting heterozygosity and epistasis.
At present, part of excellent diploid petunia varieties such as double-petal 'double waterfall' series and big flower 'fantastic' series have higher ornamental value but less pollen, less seed formation and low fertility, and the seed setting rate is further reduced by adopting a asexual ploidy way, and almost no offspring is not suitable for use; by adopting a sexual polyploidization way, the mutagen is used for treating young buds to have great damage to the development of microspores, and pollen does not exist after the pollen further reduces the flowering, so that the sexual polyploidization way is not suitable for use.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the difficulty in mutagenizing polyploids of diploid petunia with few pollen, few seeds and low fertility, the invention provides the method for cultivating the tetraploid petunia, which can improve the mutagenizing rate of the polyploids, and the cultivated tetraploid variety has wider genetic diversity, not only has good characters of diploid male parents and higher ornamental value, but also has higher seed-forming rate, and simultaneously, the method has low requirements on operation fields, instruments and equipment and relatively simple and easy operation process.
The technical scheme is as follows: a method for breeding tetraploid petunia comprises the following steps:
(1) mutagenizing axillary buds of diploid petunia plants by using a chemical inducer, and identifying and screening tetraploid branches after the mutagenized axillary buds germinate and grow into branches;
(2) taking tetraploid petunia with good fertility as a female parent, emasculating, taking out mature 2n pollen on the tetraploid branches obtained by mutagenesis, coating the pollen on the female parent column, and obtaining F1 hybrid seeds after plants are fruited;
(3) tetraploid plants are screened out in the F1 generation and are selected by a pedigree method, and the tetraploid petunia variety with excellent properties is obtained.
Some diploid petunia with less pollen, less seed formation and low fertility have gorgeous flowers and high ornamental value, but seeds are difficult to obtain, the mutagenic material is less, and the seed is hardly obtained due to lower fertility after the asexual polyploidy is mutagenized; and a sexual polyploidization way is adopted, young buds treated by a mutagen have large damage to the development of microspores, and no pollen exists after the buds bloom. Aiming at the condition that the conventional polyploid technology of the variety is insufficient, the method comprises the steps of planting diploid petunia which has high ornamental value, less pollen and less seed setting and low fertility according to the conventional technology, selecting a plant with strong growth potential as a mutagenesis material, carrying out chemical mutagenesis on axillary buds on branches to promote the tetraploid branches to have 2n pollen after blooming, and imparting the 2n pollen to tetraploid female parent posts with higher fertility to obtain F1The tetraploid variety obtained by hybridizing the seeds and performing multi-generation selection on the offspring not only has wider genetic diversity, has some excellent properties of a diploid male parent and higher ornamental value, but also has higher seed setting rate.
Before mutagenesis treatment, the diploid petunia plants are trimmed, excessive branches and weak branches on the plants are cut off, only 3-6 robust branches are reserved for each plant, and terminal buds of the branches are removed.
The diploid petunia plants are not watered or watered less 5-7 days before mutagenesis treatment to keep the soil dry so that the water content of the soil is lower than 8%, and are immediately and fully watered after mutagenesis treatment to keep the soil moist so that the water content of the soil reaches 20-25%.
After the mutagenesis treatment, the temperature is kept at 25-32 ℃, and the treatment can be carried out in open fields or plastic greenhouses in spring and summer.
The chemical inducer is colchicine, and the final mass concentration is 0.1-0.3%. The mutagenesis method comprises the following steps: covering the axilla with a medium with good liquid adsorption effect, closely contacting with the axilla, injecting the chemical inducer such as colchicine liquid medicine into the medium with a micro-syringe to soak the medium, 1 time each in the morning and afternoon every day, and continuously for 1-3 days; the mutagenized plants were shaded during mutagenesis. Preferably, the treatment concentration of colchicine is 0.2-0.3%, and the treatment days are 1-2 days; more preferably, the colchicine treatment concentration is 0.3% and the treatment days are 1 day.
After the axillary buds after the mutagenesis treatment germinate and grow into branches, tetraploid branches are screened out through morphological and cytological identification; polyploid characteristics were: compared with diploid, the leaf thickness, length and width and sepal length and width are increased, leaf surface is twisted, the volume of stigma, anther and mature pollen is increased, the diameter of petal is increased, and the edge of petal is wrinkled.
The diploid petunia is a double-petal 'double waterfall' series, a big-flower 'fantasy' series or a big-flower 'rainbow' series. The large flower "rainbow" series can be selected from Red Petunia hybrida 'U-IRIS Red', Blue Petunia hybrida 'U-IRIS Blue', Pink Petunia hybrida 'DOUBLE CASCASCADE Pink', etc.
The tetraploid petunia female parent is rose morning light.
When F1 generation is selected, F1 hybrid seeds are grown to the flowering period of an adult plant, and the ploidy of the hybrid seeds is subjected to cytological observation to identify whether the hybrid seeds are tetraploid. The main characters investigated in the pedigree selection process are as follows: the method has the advantages that excellent tetraploid varieties with stable heredity can be obtained through 3-5 generations of selection, the tetraploid varieties have wider genetic diversity, some excellent properties of diploid male parents, and high fertility and seed setting rate.
Has the advantages that:
1. the existing asexual polyploidy induction is carried out at the stage of the seed just sprouting or cotyledon, some diploid petunia with less seed setting and low fertility can be used for the mutagenesis test with less seeds and high mutagenesis death rate, the water content is difficult to control under the severe culture and induction conditions such as the dwarf of seedlings, the temperature control is strict, the seed can not be obtained almost due to lower fertility after the asexual polyploidy induction, and the tetraploid offspring can not be stored; and a sexual polyploidization way is adopted, young buds are treated by a mutagen, so that the injury to the development of microspores is large, and no pollen is generated after blooming, and the hybridization can not be carried out. Aiming at the condition that the conventional polyploid technology of the variety is insufficient, the method disclosed by the invention has the advantages that the axillary buds are induced, the number of the axillary buds on the plant is large, and a large number of repeated tests are facilitated along with the continuous formation of the plant growth. After axillary buds grow into branches, the branches with obvious polyploid characteristics and cytological identification as tetraploids are selected from the branches, 2n pollen of the branches is hybridized with the tetraploids with high fertility and high seed set to form tetraploid offspring, and then the tetraploid new variety obtained by multi-generation selection through a spectrum method not only has wide genetic diversity, has some excellent characteristics and high ornamental value of male parents, but also has high fertility and seed set rate, and better solves the polyploid mutagenesis problem of the diploid petunia variety with less pollen, less seed set, low fertility and high ornamental value. Meanwhile, the invention has low requirements on test sites (which can be carried out in open fields), detection instruments and relatively simple and easy operation process.
2. Moisture and temperature control before and after mutagenesis: the soil is kept dry (the water content of the soil is lower than 8%) without watering or with little watering 5-7 days before mutagenesis, the mitotic activity of the growth points of the axillary buds can be inhibited and slowed down, the soil is fully watered immediately after mutagenesis treatment to keep the water content of the soil moist to 20% -25%, the mitotic activity of the growth points can be accelerated when the temperature is kept between 25 ℃ and 32 ℃, more growth point meristematic cells can be in the mitotic phase due to sufficient water and proper temperature, and the mutagenesis success rate is improved.
3. Pruning treatment: the method comprises the steps of cutting excessive branches and weak branches of plants before mutagenesis, only keeping 3-6 robust branches for each plant, enabling a mutagen colchicine to have certain toxicity to the plants, if the plants are poisoned and die due to excessive branches, keeping 3-6 branches without plant death, and removing terminal buds of the branches to promote axillary buds to germinate and grow. And (3) timely removing germinated branches with unobvious polyploidy characteristics after mutagenesis, so that tetraploid branches can grow better.
4. The identification method of the invention comprises the following steps: the polyploid and diploid plants are generally huge compared with each other, after axillary buds germinate branches, agronomic characters such as leaf length and width, leaf shape index (leaf length/leaf width), leaf thickness, stem thickness, plant height, length and width of sepals, size of stigma and anther and size of pollen cells are firstly compared, and a plurality of germinated branches with obviously increased indexes are selected, so that the cytological identification workload can be reduced. When the cytological ploidy is identified, young buds are used as identification materials, the sufficient sampling materials do not influence the growth of plants, the requirement on instruments is not high, and the operation process is relatively simple and easy.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.
The cytological identification methods in the following examples are: taking young buds with the length of 2.6-5.8mm from a plant taken at 8:00-10:00 in the morning on a sunny day, fixing the young buds for 24 hours by using Carnot's fixing solution, performing gradient dehydration by using ethanol with the volume concentration of 90% and the volume concentration of 80%, and finally transferring the young buds to ethanol with the volume concentration of 70% for storage at 4 ℃ for later use; using l N hydrochloric acid 60 ℃ constant temperature water bath to flower buds for about 2-3min, washing with distilled water for 3-4 times, stripping anther from the flower buds, dyeing with 1% acetic acid carmine by mass concentration, conventionally dyeing and tabletting, copying the slices by flame for 2s for color separation, counting chromosomes of pollen mother cells in meiosis stage under 100 times of oil lens, wherein the chromosome number of tetraploid gamete is n-2 x-14; wherein the Carnot fixing solution is prepared from absolute ethyl alcohol and glacial acetic acid according to the volume ratio of 3: 1. The volume is hundred1% (mass concentration) prepared by 45% acetic acid solutionMagenta inkThe preparation method of the dyeing liquid comprises the following steps: adding 45ml of acetic acid and then 55ml of water, and uniformly mixingMagenta inkSlowly pouring 1 g of the powder into 100ml of 45% acetic acid solution, boiling while stirring, boiling, cooling and filtering for use.
Example 1: breeding method of tetraploid petunia
1) Taking Red Petunia hybrida 'U-IRIS Red' in the large-flower diploid Petunia rainbow series as a test object, and sowing and planting according to a conventional technology;
2) and (3) cutting excessive branches and weak and small branches from the plants in adult plants, only keeping 3 robust branches for each plant, removing terminal buds of the branches, not watering or watering little before mutagenesis for 5 days to keep the soil dry to enable the water content of the soil to be lower than 8%, immediately and fully watering after mutagenesis treatment to keep the soil moist to enable the water content of the soil to reach 20%, and keeping the temperature at about 25 ℃. The axillary buds on the plants are induced by a chemical inducer, wherein the chemical inducer is colchicine, and the final mass concentration is 0.1%. The mutagenesis method comprises the following steps: covering absorbent cotton at axilla to make it closely contact with the axilla, injecting colchicine liquid into medium with micro-syringe to soak absorbent cotton, 1 time each in the morning and afternoon every day, and continuously for 3 days; the mutagenized plants were shaded during mutagenesis.
3) After mutagenesis, axillary buds germinate and grow into branches, the branches with polyploidy characteristics, such as thickened leaves, distorted leaves, enlarged flower stigma, enlarged sepals and the like, are selected, and the buds on the branches are subjected to cytological identification to be determined as tetraploids; and (3) timely removing germinated branches with unobvious polyploidy characteristics after mutagenesis, so that tetraploid branches can grow better.
4) Selecting a tetraploid variety 'rose morning light' with higher fertility as a female parent, performing castration treatment before hybridization, taking out mature 2n pollen on the mutagenized tetraploid branches, coating the pollen on the female parent column, and obtaining F1 hybrid seeds after plants are fruited;
5) growing the F1 hybrid seed to the flowering phase, and taking the young bud for cytological identification to determine the young bud as a tetraploid plant;
6) the F1 progeny were selected by pedigree (the main traits examined in the pedigree selection process were: petal color, petal size and shape, plant disease resistance and seed setting rate), and through 3 generations of selection, excellent tetraploid variety with wide genetic diversity, excellent diploid male parent character and high seed setting rate may be obtained, with each fruit containing over 90 seeds and the original male parent containing less than 15 seeds.
Example 2: breeding method of tetraploid petunia
1) Taking Blue Petunia hybrida 'U-IRIS Blue' in the large-flower diploid Petunia rainbow series as a test object, and sowing and planting according to a conventional technology;
2) and (3) cutting excessive branches and weak and small branches on plants in adult plants, only keeping 4 robust branches for each plant, removing terminal buds of the branches, and keeping the soil dry by not watering or watering a little 6 days before mutagenesis to ensure that the water content of the soil is lower than 8%. Immediately after mutagenesis treatment, the soil is watered fully to keep the soil moist so that the water content of the soil reaches 22 percent, and the temperature is kept at about 30 ℃. The axillary buds on the plants are induced by a chemical inducer, wherein the chemical inducer is colchicine, and the final mass concentration is 0.2%. The mutagenesis method comprises the following steps: covering absorbent cotton at axilla to make it closely contact with the axilla, injecting colchicine liquid into medium with micro-syringe to soak absorbent cotton, 1 time each in the morning and afternoon every day, and continuously for 2 days; the mutagenized plants were shaded during mutagenesis.
3) After mutagenesis, axillary buds germinate and grow into branches, the branches with polyploidy characteristics, such as thickened leaves, distorted leaves, enlarged flower stigma, enlarged sepals and the like, are selected, and the buds on the branches are subjected to cytological identification to be determined as tetraploids; and (3) timely removing germinated branches with unobvious polyploidy characteristics after mutagenesis, so that tetraploid branches can grow better.
4) Selecting a tetraploid variety 'rose morning light' with higher fertility as a female parent, performing castration treatment before hybridization, taking out mature 2n pollen on the mutagenized tetraploid branches, coating the pollen on the female parent column, and obtaining F1 hybrid seeds after plants are fruited;
5) growing the F1 hybrid seed to the flowering phase, and taking the young bud for cytological identification to determine the young bud as a tetraploid plant;
6) the F1 progeny were selected by pedigree (the main traits examined in the pedigree selection process were: petal color, petal size and shape, plant disease resistance and seed setting rate), and excellent tetraploid variety with stable heredity can be obtained through 4 generations of selection, wherein the tetraploid variety has wider genetic diversity, some excellent properties of a diploid male parent and higher seed setting rate, the number of seeds contained in each fruit exceeds 85, and the number of seeds contained in each fruit of an original male parent is less than 20.
Example 3: breeding method of tetraploid petunia
1) Taking Pink Petunia hybrida 'DOUBLE CASCASCADE Pink' in the DOUBLE-waterfall series of DOUBLE-petal diploid Petunia as a test object, and sowing and planting according to a conventional technology;
2) and (3) cutting excessive branches and weak and small branches on plants in adult plants, only keeping 5 robust branches for each plant, removing terminal buds of the branches, and keeping the soil dry by not watering or watering a little 7 days before mutagenesis to ensure that the water content of the soil is lower than 8%. Immediately after mutagenesis treatment, the soil is fully watered to keep the soil moist so that the water content of the soil reaches 25 percent, and the temperature is kept at 32 ℃. The axillary buds on the plants are induced by a chemical inducer, wherein the chemical inducer is colchicine, and the final mass concentration is 0.3%. The mutagenesis method comprises the following steps: covering absorbent cotton at axilla to make it closely contact with the axilla, injecting colchicine liquid into medium with micro-syringe to soak absorbent cotton, 1 time each in the morning and afternoon every day, and continuously for 1 day; the mutagenized plants were shaded during mutagenesis.
3) After mutagenesis, axillary buds germinate and grow into branches, the branches with polyploidy characteristics, such as thickened leaves, distorted leaves, enlarged flower stigma, enlarged sepals and the like, are selected, and the buds on the branches are subjected to cytological identification to be determined as tetraploids; and (3) timely removing germinated branches with unobvious polyploidy characteristics after mutagenesis, so that tetraploid branches can grow better.
4) Selecting a tetraploid variety 'rose morning light' with higher fertility as a female parent, performing castration treatment before hybridization, taking out mature 2n pollen on the mutagenized tetraploid branches, coating the pollen on the female parent column, and obtaining F1 hybrid seeds after plants are fruited;
5) growing the F1 hybrid seed to the flowering phase, and taking the young bud for cytological identification to determine the young bud as a tetraploid plant;
6) the F1 progeny were selected by pedigree (the main traits examined in the pedigree selection process were: petal color, petal size and shape, plant disease resistance and seed setting rate), and excellent tetraploid variety with stable heredity can be obtained through 5 generations of selection, wherein the tetraploid variety has wider genetic diversity, some excellent characters of diploid male parent and higher seed setting rate, the number of seeds contained in each fruit exceeds 30, and the number of seeds contained in each fruit of original male parent is less than 5.
Example 4
This example examines the comparison of the mutagenic effect of different colchicine concentrations, treatment days and control of moisture before mutagenesis, and the procedure is the same as in example 1 except that the factors referred to in Table 1 are varied.
TABLE 1 comparison of the mutagenic effect of the control of moisture before mutagenesis and different colchicine concentrations, treatment days
In the above table, moisture control prior to mutagenesis means that 5-7 days prior to mutagenesis, no or little water is applied to keep the soil dry to a moisture content of less than 8%. Immediately and fully watering after mutagenesis treatment to keep the soil moist to ensure that the soil moisture content reaches 20% -25%, and the non-control means that the soil is always moist and the soil moisture content reaches 20% -25%.
As can be seen from Table 1, the control of water content before mutagenesis can effectively improve the induction rate, in addition, the concentration of colchicine and the treatment days also have influence on the induction rate, the concentration of colchicine is 0.2-0.3%, the best tetraploid induction rate can be obtained after 1-2 days of treatment, and the induction rate can reach 23.3-26.6%.
Claims (1)
1. A method for cultivating tetraploid petunias is characterized by comprising the following steps:
(1) mutagenizing axillary buds of diploid petunia plants by using a chemical inducer, pruning the diploid petunia plants before mutagenizing, cutting off excessive branches and weak branches on the plants, only keeping 3-6 robust branches of each plant, and removing branch top buds; controlling the water content of the cultivating soil of the diploid petunia plants to be lower than 8% 5-7 days before mutagenesis treatment, and immediately and fully watering after mutagenesis treatment to enable the water content of the soil to reach 20% -25%; keeping the soil moist; the temperature is kept at 25-32 ℃; after the axillary buds after the mutagenesis treatment germinate and grow into branches, tetraploid branches are screened out through morphological and cytological identification; polyploid characteristics were: compared with the diploid, the thickness, the length and the width of the leaves and the length and the width of the sepals are increased, the leaves are twisted, the volumes of stigma, anther and mature pollen are increased, the diameter of petals is increased, and the edges of the petals are wrinkled; the chemical inducer is colchicine, and the final mass concentration is 0.1-0.3%; the diploid petunia is a double-petal 'double-waterfall' series or a large-flower 'rainbow' series;
(2) the tetraploid petunia with good fertility performance is taken as a female parent, and the tetraploid petunia female parent is rose morning light; after castration, taking out mature 2n pollen on the tetraploid branches obtained by mutagenesis, coating the pollen on the head of a female parent, and obtaining F1 hybrid seeds after plants are fruited;
(3) selecting tetraploid plants from the F1 generation for pedigree selection to obtain tetraploid petunia variety with excellent properties; when F1 generation is selected, F1 hybrid seeds are grown to the flowering period of an adult plant, and the ploidy of the hybrid seeds is subjected to cytological observation to identify whether the hybrid seeds are tetraploid.
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